All metabolic pathways must be regulated in order to serve the needs of individual cells, organs, or the whole body. Regulation of the metabolic pathways that provide fuel molecules, for example, carbohydrates, is essential if the supply is to be maintained in the various nutritional, metabolic, and pathologic conditions that are encountered in vivo. Metabolic fuel regulation involves provision of the specific fuel needs of each tissue, including making alternative fuels available. It also involves the transport of various involved substrates throughout the body, together with mechanisms to control their concentration in the blood.
These mechanisms ensure a continuous supply of glucose between meals and during a fast. Many conditions, typically associated with an enzyme deficiency, result in low blood glucose (hypoglycemia). Other, pathologic enzyme deficiencies can cause different but equally serious changes in carbohydrate metabolism, for example, insulin deficiency, which results in diabetes mellitus and increased blood glucose (hyperglycemia).
Glucose is know to be the primary currency of metabolic energy, and it circulates in the bloodstream to all the tissues and organs in the body. In the resting state humans typically utilize about 10 grams of glucose per hour, sixty percent of which goes to the brain. In the active state, the brain continues to draw about six grams of glucose per hour, but muscle use of glucose jumps to as much as forty grams per hour.
At mealtime, the consumption of food followed by its metabolism results in an infusion of glucose into the circulation at a rate greater than that required by the brain and other organs and tissues. To prevent an unacceptable rise in blood glucose level, i.e., hyperglycemia, glucose is extracted from circulation and stored as glycogen, principally in muscle tissue. The resulting process, which is regulated by insulin, is termed "insulin-stimulated glucose uptake."
In response to rising blood glucose levels and other stimuli related to food consumption, insulin is secreted by the pancreas into the bloodstream. Insulin is a protein hormone produced by the beta cells of the pancreatic Islets of Langerhans. Insulin decreases blood glucose in two ways. First, it signals muscle and fat tissues (so-called "peripheral" tissues) to increase glucose uptake for storage, respectively, as glycogen and fat. Second, insulin signals the liver to reduce glucose secretion.
During exercise, muscle demand for energy increases dramatically. Initially, muscles draw on their internal glycogen stores until this supply of glucose is exhausted. Glucose is also released into circulation by the liver as needed by the brain and non-muscle tissues. The process of drawing on liver glucose stores, which is also mediated by the pancreas, is termed "glucagon-stimulated glucose secretion".
In response to falling blood glucose levels related to vigorous activity, glucagon is secreted by the pancreas into the bloodstream. Glucagon is a polypeptide hormone produced by the alpha cells of the Islets of Langerhans in the pancreas. Glucagon increases blood glucose principally by stimulating glycogen breakdown to glucose, and subsequent secretion of that glucose, by the liver. It will be understood, then, that a major function of the liver is to maintain a relatively constant level of glucose in the blood.
The processes of carbohydrate metabolism include the pathways of glycolysis (under both aerobic and anaerobic conditions), oxidation of pyruvate to acetyl-CoA, glycogen biosynthesis in both muscle and liver, glycogen degradation in both muscle and liver, gluconeogenic pathways in both liver and kidney, the pentose phosphate pathway, the uronic acid pathway, and pathways relating to the metabolism of fructose, sorbitol (polyol), galactose, and amino sugars (hexosamines). There are dozens of enzymes implicated in some of these pathways, and such enzymes include glucokinase, the glycogen synthase system enzymes, phosphofructo-kinase-1, pyruvate kinase, and pyruvate dehydrogenase (enzymes of glycolysis and glycogenesis), pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-biphosphatase, and glucose-6-phosphatase (enzymes of gluconeogenesis), and glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase (enzymes of the pentose phosphate pathway and lipogenesis).
To this day, however, many underlying mechanisms of fuel metabolism remain confusing and the subject of academic controversy. Over the years, for example, views on whole-body glucose metabolism and the mechanisms of repletion of liver glycogen during the fasted-to-fed transition have continued to shift. McGarry, J. D., et al., Ann. Rev. Nutr. 7:51-73 (1987). As described herein, identification by Cooper of a third pancreatic hormone, amylin, has added a further factor to the already complicated fuel metabolism picture, and has necessitated a reevaluation of both fuel pathways and fuel pathway mechanisms. Eg., Cooper, G. J. S., et al., Biochim. Biophys. Acta 1014:247-252 (1989). Certain of these mechanisms, however, are well established.
Glycogen is understood to be an important factor in the generation and storage of metabolic energy, it being a readily mobilized storage form of glucose. Glycogen is a very large, branched polymer of glucose residues, most of which are linked by .alpha.-1,4-glycosidic bonds. The branches are created by an .alpha.-1,6 linkage between two glucose units. The two major sites of glycogen storage are the liver and skeletal muscle. The concentration of glycogen is higher in the liver, but more total glycogen is stored in skeletal muscle because of its greater mass.
As indicated, the synthesis and degradation of glycogen are important because they are used by the body to regulate the blood glucose level, glycogen providing a reservoir of glucose for use during strenuous muscle activity. The function of muscle glycogen is primarily to act as a readily available source of hexose units for glycolysis within the muscle itself. Liver glycogen is largely concerned with the export of hexose units for the maintenance of blood glucose levels, particularly between meals. It is known that glycogen synthesis and degradation occur through distinct reaction pathways. As in many other biological systems, the enzymes of glycogen metabolism are regulated by reversible phosphorylation.
FIG. 1 shows that regulation of glycogen metabolism is effected by a balance in activities between glycogen synthase and glycogen phosphorylase, which are under substrate control (through allostery) as well as hormonal control. Skeletal muscle phosphorylase exists in two interconvertible forms. Phosphorylase a is active and phosphorylase b is normally less active. Phosphorylase b is converted to phosphorylase a by phosphorylation of a serine residue in each of two subunits of the molecule. Hormones such as epinephrine and glucagon bind to receptors in the plasma membrane of target cells and trigger the activation of adenylate cyclase. Adenylate cyclase in the plasma membrane catalyzes the formation of cyclic AMP from ATP. The increased intracellular level of cyclic AMP activates a protein kinase, which is inactive in the absence of cyclic AMP. The protein kinase phosphorylates both phosphorylase kinase and glycogen synthase. The phosphorylation of these enzymes is the basis for coordinated regulation of glycogen synthesis and breakdown.
Glycogenolysis can be terminated and glycogenesis can be stimulated synchronously, or vice versa, because both processes are keyed to the activity of cAMP-dependent protein kinase. Both phosphorylase kinase and glycogen synthase may be reversibly phosphorylated in more than one site by separate kinases and phosphatases. These secondary phosphorylations modify the sensitivity of the primary sites to phosphorylation and dephosphorylation. Phosphorylation by the cyclic-AMP-dependent protein kinase switches on phosphorylase (by activating phosphorylase kinase) and simultaneously switches off glycogen synthase (directly) by converting it to its inactive form. Thus, inhibition of glycogen breakdown (glycogenolysis) enhances net glycogen synthesis (glycogenesis), and inhibition of glycogenesis enhances net glycogenolysis. Of further significance in the regulation of glycogen metabolism is the finding that the dephosphorylation of phosphorylase a, phosphorylase kinase, and glycogen synthase b (D-form) is accomplished by a single enzyme of wide specificity known as protein phosphatase-1. In turn, protein phosphatase-1 is inhibited by cAMP-dependent protein kinase via inhibitor-1. FIG. 1 also shows the control of glycogenolysis and glycogenesis by cAMP-dependent protein kinase. The reactions that lead to glycogenolysis as a result of an increase in cAMP concentrations include the conversion of glycogen synthase a to glycogen synthase b, the conversion of phosphorylase kinase b to phosphorylase kinase a, and the conversion of glycogen phosphorylase b to glycogen phosphorylase a. Concomitantly, the opposite conversions are inhibited under these conditions. The reverse occurs when cAMP concentrations decrease as a result of phosphodiesterase activity, leading to glycogenesis. For a discussion of carbohydrate fuel metabolism including glycolysis, gluconeogenesis, glycogenolysis and glycogen synthesis, see, e.g., McGarry, J. D., et al., supra; L. Stryer, Biochemistry (3d Edition 1988).
In 1929 Cori and Cori proposed that glucose carbons could be cycled in mammals through the sequence: liver glycogen.fwdarw.blood glucose.fwdarw.muscle glycogen.fwdarw.blood lactic acid.fwdarw.liver glycogen. Cori, C. F. and Cori, G. T., J. Biol. Chem. 81:389-403 (1929). For its part, muscle glycogen catabolism proceeds as follows. Glycogen is cleaved in the presence of glycogen phosphorylase a and orthophosphate to yield a phosphorylated sugar, glucose-1-phosphate. The enzyme glycogen phosphorylase a facilitates the sequential removal of glycosyl residues from the nonreducing end of the glycogen molecule, in which the glycosidic linkage between C-1 of the terminal residue and C-4 of the adjacent residue is split. The glucose 1-phosphate formed in the phosphorolytic cleavage of glycogen is converted into glucose 6-phosphate by another enzyme, phosphoglucomutase.
FIG. 2 shows the key enzymes in the control of glycolysis, gluconeogenesis, and glycogen metabolism in liver and muscle, the major differences in the muscle and liver pathways being that muscle is not affected by glucagon and does not contain the enzyme glucose-6-phosphatase, both of which are part of the liver pathways where indicated in the Figure. Glucose 6-phosphatase is a hydrolytic enzyme that enables glucose to leave the liver. It catalyzes the formation of glucose from glucose-6-phosphate, whereupon glucose can exit the liver cell via glucose transporters. Glucose-6-phosphate cannot be transported. The glucose-6-phosphatase enzyme is absent from the brain as well as muscle. The net result is that glucose 6-phosphate is retained by muscle and brain, which need large amounts of this fuel for the generation of ATP. By contrast, glucose is not a major fuel for the liver, an "altruistic" organ which stores and releases glucose primarily for the benefit of other tissues.
Glycogen metabolism is profoundly affected by specific hormones. While the mechanism of insulin action is not yet fully understood, insulin is known to increase the capacity of the liver to synthesize glycogen. Insulin also accelerates glycolysis in the liver, which in turn increases the synthesis of fatty acids. Whether liver glycolysis is the major or predominant source of pyruvate for fatty acid synthesis, however, has not been established. The entry of glucose into muscle and adipose cells is also promoted by insulin. The hormones epinephrine and glucagon have certain effects that counteract those of insulin. Muscular activity or the anticipation of muscular activity leads to the release of epinephrine from the adrenal medulla. Epinephrine markedly stimulates glycogen breakdown in muscle and, to a lesser extent, in liver. Another action of this catecholamine hormone is to inhibit the uptake of glucose by muscle. Instead, fatty acids released from adipose tissue are used as fuel. Epinephrine also increases the amount of glucose released into the blood by the liver and decreases the utilization of glucose by muscle by, respectively, stimulating the secretion of glucagon and inhibiting the secretion of insulin.
As noted above, the liver is responsive to glucagon, a polypeptide hormone that is secreted by the a cells of the pancreas when the blood sugar level is low. Glucagon increases the blood sugar level by stimulating the breakdown of glycogen in the liver and at the same time inhibiting glycogen synthesis. The net result of these actions is to markedly increase the output of liver glucose.
A newly discovered pancreatic hormone, amylin, is expressed mainly in pancreatic .beta. cells and co-secreted with insulin. Amylin was first discovered as the major protein constituent of the islet amyloid which is found in patients with type 2 diabetes mellitus. E.g. Cooper, G. J. S., et al., Proc. Nat. Acad. Sci. USA 84-8628:8632 (1987); Cooper, G. J. S., et al, Biochim. Byophys. Acta 1014:247-252 (1989). Human amylin has a somewhat unusual amino acid composition in that it contains no acidic residues. Amylin is a 37 amino acid peptide having two post translational modifications, a Cys.sup.2 -Cys.sup.7 intramolecular disulfide bond and a carboxy-terminal amide group. It was also discovered by Cooper that the presence of both of these post-translational modifications in the peptide structure of the synthetic molecule yield the greatest biological activity. E.g. Cooper, G. J. S., et al., Proc. Natl. Acad. Sci. U.S.A. 84:8628-8632 (1987); Cooper, G. J. S., et al. in Diabetes 1988, ed. Larkins, R., Zimmet, P. & Chisholm, D. (Elsevier, Amsterdam), pp. 493-496 (1989).
Human amylin has 43-46% sequence identity with human CGRP-1 and CGRP-2 (calcitonin gene-related peptides 1 and 2, respectively). Human amylin also has weaker sequence similarities with insulin, the relaxins, and the insulin-like growth factors (IGFs). This observation concerning sequence similarities supports the determination that there is a peptide hormone superfamily which includes the CGRPs, amylin, and the A-chain related region of the relaxins, insulin and the IGFs. Cooper, G. J. S., et al., Progress in Growth Factor Research 1:99-105 (1989).
Amylin is the product of a single gene present on chromosome 12 in humans. This gene has typical features of one encoding a polypeptide hormone, including prepro- and proamylin sequences, typical 5' and 3' dibasic processing signals, and a Gly residue 3' to the codon for the carboxyterminal Tyr, which constitutes an amidation signal. Roberts, A. N., et al., Proc. Nat. Acad. Sci. U.S.A. 86:9662-9666 (1989). There is a high degree of interspecies conservation between both the amylins and the CGRPS, particularly in the region of the amino- and carboxy- termini. These regions of strong conservation correspond to the structural regions within the molecules which contain the post-translational modifications necessary for at least some of their biological activities. The variable sequence in the mid-portion of the amylin molecule contains the region said to be primarily responsible for amyloid formation.
Amylin is synthesized in the islets (Leffert, J. D., et al., Proc. Natl. Acad. Sci. U.S.A. 86:3127-3130 (1989) and Roberts, A. N., et al., Proc. Natl. Acad. Sci. U.S.A. 86:9662-9666 (1989)), from which it is secreted along with insulin in response to nutrient secretagogues. Ogawa, A., et al., J. Clin. Invest. 85:973-976 (1990). Amylin is packaged in the .beta.-cell secretory granules, along with insulin. Experiments using an isolated perfused rat pancreas indicate that both glucose and arginine can stimulate amylin secretion in a biphasic pattern similar to that seen with insulin. Additionally, as with insulin, amylin secretion is amplified by combining the two secretagogues. Ogawa et al. J. Clin. Inves. 85:973-976 (1990); Fehmann et al., FEBS Letters 262:279-281 (1990). The amylin protein content within the pancreas has not been defined with certainty, although estimates using the rat pancreas as a model indicate the amylin mass to be about 4 to 6 times that of glucagon and 1 to 2 times of that of somatostatin.
Deposition of islet amyloid correlates well with the loss of islet .beta.-cells and defective insulin secretion found in type 2 diabetics. Gepts, W., The Islets of Lanaerhans, ed. Cooperstein, S. J. & Watkins, D. (Academic Press, New York, N.Y.), pp. 321-356 (1980), Fehmann, H. C., et al., FEBS Lett. 262:279-281 (1990); Cooper, G. J. S., et al., Biochim. Biophys. Acta 1014:247-258 (1989). The ability of amylin to cause insulin resistance in many model systems, combined with its presence in human islet amyloid in diabetic pancreases supports the determination that it is central to the pathogenesis of non-insulin dependent diabetes mellitus. E.g. Cooper, G. J. S., et al., Biochim. Biophys. Acta 1014:247-258 (1989); Leighton, B. & Cooper, G. J. S., Nature (Lond) 335:632-635 (1988). Amylin also has been reported to produce marked effects on glucose metabolism in animals in vivo. In experiments utilizing the euglycemic, hyperinsulinemic glucose clamp, amylin reversed insulin-mediated suppression of hepatic glucose output in rats. Molina, J. M., Cooper, G. J. S., Leighton B. & Olefsky, J. M., Diabetes 39:260-265 (1990) and Koopmans, S. J., et al., Diabetes 39:101A (1990). Amylin also decreased peripheral uptake of glucose. Molina, J. M., Cooper, G. J. S., Leighton, B. & Olefsky, J. M., Diabetes 39:260-265 (1990); Koopmans, S. J., et al., Diabetes 39:101A (1990); Young, D. A., et al., Diabetes 39 (Suppl. 1):116A (1990).
Plasma lactate has long been known to be the principal three-carbon substrate for gluconeogenesis and for fatty acid synthesis. Other important three-carbon substrates include alanine and glycerol. As such, lactate is now considered by some to be a key link in the pathways that lead to storage of glycogen in liver and to storage of triglyceride in fat cells. Others have viewed lactate principally as an end-product of glycolysis, which supplies energy in the form of ATP under anaerobic conditions and distributes the metabolic load over both space and time. For example, Stryer, supra, identifies lactate as a dead end in metabolism, the only purpose of the reduction of pyruvate to lactate being to regenerate NAD.sup.+ so that glycolysis can proceed in active skeletal muscle and erythrocytes. In other words, as described above with regard to the Cori cycle, the liver is believed to furnish glucose to the contracting skeletal muscle, which derives ATP from the glycolytic conversion of glucose into lactate, glucose then being synthesized from lactate by the liver.
The principal source of the lactate which enters the Cori Cycle has remained the subject of debate. While some indicate that the lactate originates from muscle in diabetic subjects (Capaldo, B., et al., J. Clin. Endo. Metab. 71:1220-1223 (1990)), others say that it comes from tissues other than muscle, such as fat. Jansson, P. A., et al., Diaetalogia, 33:253-256 (1990). Whatever its source, still another group also concluded that, while increased substrate delivery to the liver and increased efficiency of intrahepatic substrate conversion to glucose are both important factors for the increased gluconeogenesis characteristic of Type 2 diabetics, tissues other than muscle are responsible for the increased delivery of gluconeogenic precursors to the liver. Consoli et al., J. Clin. Invest. 86:2038-2045 (December 1990).
The role of amylin also remains the subject of debate. In skeletal muscle in vitro, amylin has been discussed in regard to or implicated in many different pathways of carbohydrate metabolism, including incorporation of glucose into glycogen (Leighton, B. & Cooper, G. J. S., Nature 335:632-635 (1988); Cooper, G. J. S., et al., Proc. Natl. Acad. Sci. U.S.A. 85:7763-7766 (1988); Leighton, B., and Foot, E., Biochem J. 269:19-23 (1990)), glycogenolysis (Young et al., Am J. Physiol. 259:457-461 (1990); Leighton, B., Foot, E. A. & Cooper, G. J. S. (1989) Diab. Med. 6: Suppl. 2, A4 (1989)), glycogenesis (Young et al., supra), and glucose uptake (Young et al., supra; Ciaraldi, T. P., Cooper, G. J. S. & Stolpe, M., Diabetes 39, 149A (1990); Kreutter, D. et al., Diabetes 39, (Suppl. 1) :121A (1990); Leighton, B., et al., FEBS Letters 249:357-361 (1989)). The effects of amylin in skeletal muscle depend upon distribution of fiber type. Leighton, B., Foot, E. A. & Cooper, G. J. S. (1989) Diab. Med. 6 (Supp. 2):A4 (1989). While amylin was reported to inhibit glycogen synthesis in both red (soleus) and white (extensor digitorum longus) muscle in vitro, it was reported to stimulate glycogenolysis (and subsequent lactate production) only in white muscle. Id. White (type II) muscle fibers constitute the bulk of muscle mass in most mammals surveyed. Ariano, M. A., et al., J. Histochem. Cytochem. 21:51-55 (1973). The effects of amylin on glycogen synthesis in isolated red muscle (soleus) were reported equipotent with those of the pure .beta.-adrenergic agonist, isoprenaline. Leighton, B. & Cooper, G. J. S., Nature (Lond) 335:632-635 (1988). In L6 myocytes, maximal reduction of glucose uptake has been reported at 10 pM. Ciaraldi, T. P., Cooper, G. J. S., & Stolpe, M., Diabetes 39:149A (1990); Kreutter, D., et al., Diabetes 39 (Suppl. 1):121A (1990).
Amylin Corporation's International Patent Application No. PCT/US89/00049, "Treatment of Type 2 Diabetes Mellitus" was published on Jul. 13, 1989, bearing International Publication Number WO 89/06135. The inventions described therein by Cooper and Greene include compounds and methods for blocking or mitigating the effects of amylin, which enables, for example, the treatment of type 2 diabetics. Type 2 diabetes is characterized by insulin resistance, which may be defined as a failure of the normal metabolic response of peripheral tissues to the action of insulin. In clinical terms, insulin resistance is present when normal or elevated blood glucose levels persist in the face of normal or elevated levels of insulin. It represents, in essence, a glycogen synthesis inhibition, by which either basal or insulin-stimulated glycogen synthesis, or both, are reduced below normal levels.
Application PCT/US89/00049 describes and claims means to accomplish amylin regulation, for example, by blocking the binding of amylin, calcitonin gene related peptide (CGRP), and other amylin agonists by the use of competitive inhibitors including substituted or altered peptides or subpeptides of amylin or CGRP, or by regulation of the expression or production or release of amylin or CGRP. Chemical antagonists to amylin which bind to the amylin receptor without triggering a response are used to reduce the effects of amylin or amylin agonists which act to inhibit the body's basal and insulin-stimulated responses to glucose, or to prevent the interference of those molecules with insulin release.
The application also sets forth methods for identifying additional compounds having utility for the treatment of type 2 diabetes. In this regard, the application describes the use of biological screening for synthetic or other amylin antagonists. For example, a potential or suspected antagonist is added to isolated muscle or muscle cells together with purified amylin, in the presence or absence of insulin, and glucose uptake by cells in the tissue culture is monitored. An increase in the uptake in the presence of a potential or suspected antagonist is relied upon to indicate that the compound had the required inhibitory properties. The application also discloses the use of isolated hepatocytes, islets of Langerhans or isolated islet B cells in a similar protocol in which increased insulin output is monitored instead. The application also discloses immunoassay-type screening in which the ability of test samples containing one or more synthetic or other compounds to displace amylin or anti-idiotype antibodies from monoclonal antibodies immobilized in microtitre plates is used to screen for materials which should be further evaluated under the biological testing parameters noted above.
Of great utility would be a further functional assay system or systems in which a potential or suspected agonist or antagonist of amylin could not only be identified, but characterized and specifically evaluated based upon its ability to stimulate or inhibit amylin activity at its major site or sites of action and independent of another modulator of cell action, that is, without needing to measure inhibition of an insulin-stimulated process. This site of action has now been discovered, and such novel assay systems has been invented and are described and claimed herein.
Other surprising and important aspects of amylin action in vivo on carbohydrate metabolism have also now been discovered and are described herein as the basis of further novel assay systems. First, as set forth in the below Examples, we have discovered that amylin acts primarily in vivo to increase plasma lactate levels, not glucose levels as originally believed, and in fasted animals that the increased lactate then results in sharply increased plasma glucose levels. More specifically, in lightly anesthetized rats which were fasted for 18 hours to deplete their stores of hepatic glycogen, amylin injections stimulated lactate production. These rises in plasma lactate were followed about 10 to 30 minutes later by increased plasma glucose levels. Importantly, these effects were observed for both intravenous and subcutaneous injections. The effects of amylin in fed rats differ from effects in fasted animals. In fed rats with presumably normal liver glycogen stores, amylin causes the same marked rise in plasma lactate; however, this lactate rise is followed by only a modest or no rise in plasma glucose. Glucagon is also known to increase plasma glucose, as described above, an action which reflects the important counterregulatory role of glucagon in preventing hypoglycemia. In both fasted and fed rats, however, while amylin produces a sharp increase in plasma lactate, glucagon exerted no effect on plasma lactate levels. Amylin was also discovered to cause greater increases in plasma glucose than glucagon in fasted rats, while these relative activities are reversed in fed rats.
We have discovered that amylin is an anabolic hormonal partner for insulin. Amylin directly stimulates the supply of 3-carbon substrate for gluconeogenesis, a principle avenue to hepatic glycogen synthesis, and in a dose-dependent fashion. Amylin also reduces skeletal muscle insulin sensitivity without affecting insulin-stimulated glucose uptake in fat cells, and increases the supply of 3-carbon substrates for fatty acid synthesis in the liver. These discoveries provide a basis for additional assay systems which evaluate the ability of a known or suspected agonist or antagonist of amylin function to affect amylin action, and to generate dose response profiles for said known or suspected agonists or antagonists which may then, optionally, be contrasted with dose response profiles prepared in positive and/or negative control assays.